Methods for Quantification of in vivo Changes in Protein Ubiquitination following Proteasome and Deubiquitinase Inhibition

Broad Institute of MIT and Harvard, 7 Cambridge Center, Cambridge, Massachusetts 02142, USA.
Molecular & Cellular Proteomics (Impact Factor: 6.56). 04/2012; 11(5):148-59. DOI: 10.1074/mcp.M111.016857
Source: PubMed


Ubiquitination plays a key role in protein degradation and signal transduction. Ubiquitin is a small protein modifier that is adducted to lysine residues by the combined function of E1, E2, and E3 enzymes and is removed by deubiquitinating enzymes. Characterization of ubiquitination sites is important for understanding the role of this modification in cellular processes and disease. However, until recently, large-scale characterization of endogenous ubiquitination sites has been hampered by the lack of efficient enrichment techniques. The introduction of antibodies that specifically recognize peptides with lysine residues that harbor a di-glycine remnant (K-ε-GG) following tryptic digestion has dramatically improved the ability to enrich and identify ubiquitination sites from cellular lysates. We used this enrichment technique to study the effects of proteasome inhibition by MG-132 and deubiquitinase inhibition by PR-619 on ubiquitination sites in human Jurkat cells by quantitative high performance mass spectrometry. Minimal fractionation of digested lysates prior to immunoaffinity enrichment increased the yield of K-ε-GG peptides three- to fourfold resulting in detection of up to ~3300 distinct K-GG peptides in SILAC triple encoded experiments starting from 5 mg of protein per label state. In total, we identify 5533 distinct K-ε-GG peptides of which 4907 were quantified in this study, demonstrating that the strategy presented is a practical approach to perturbational studies in cell systems. We found that proteasome inhibition by MG-132 and deubiquitinase inhibition by PR-619 induces significant changes to the ubiquitin landscape, but that not all ubiquitination sites regulated by MG-132 and PR-619 are likely substrates for the ubiquitin-proteasome system. Additionally, we find that the proteasome and deubiquitinase inhibitors studied induced only minor changes in protein expression levels regardless of the extent of regulation induced at the ubiquitin site level. We attribute this finding to the low stoichiometry of the majority ubiquitination sites identified in this study.

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Available from: Karl R Clauser, Jan 28, 2016
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    • "This remnant causes a distinct mass shift of the peptide mass that can be used to precisely identify and localize the site of ubiquitination in the peptide. The recent development of specific antibodies directed against diglycinemodified peptides enables the efficient isolation of these peptides and the identification of ubiquitination sites by MS [31] [32] [33] [34] [35]. Because of the denaturing step that is necessary before trypsin digestion , the identification of nonubiquitinated interactors using this approach is minimal. "
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    ABSTRACT: Protein ubiquitination plays an important role in the regulation of many cellular processes, including protein degradation, cell cycle regulation, apoptosis and DNA repair. To study the ubiquitin proteome we have established an immunoaffinity purification method for the proteomic analysis of endogenously ubiquitinated protein complexes. A strong, specific enrichment of ubiquitinated factors was achieved using the FK2 antibody bound to protein G beaded agarose, which recognizes mono-ubiquitinated and poly-ubiquitinated conjugates. Mass spectrometric (MS) analysis of two FK2 IPs resulted in the identification of 296 FK2-specific proteins in both experiments. The isolation of ubiquitinated and ubiquitination-related proteins was confirmed by pathway analyses (using IPA and GO-annotation enrichment). Additionally, comparing the proteins that specifically came down in the FK2 IP with databases of ubiquitinated proteins showed that a high percentage of proteins in our enriched fraction was indeed ubiquitinated. Finally, assessment of protein-protein interactions revealed that significantly more FK2-specific proteins were residing in protein complexes than in random protein sets. This method, which is capable of isolating both endogenously ubiquitinated proteins as well as their interacting proteins, can be widely used for unravelling ubiquitin-mediated protein regulation in various cell systems and tissues when comparing different cellular states.
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    • "In fact, Festa et al. (2010) identified 55 pupylation sites from a single sample in Mycobacterium tuberculosis (Mtb). As the further improvement of MS techniques, the higher-throughput identification of K-GG peptides was achieved by the direct enrichment of K-GG Peptides in vivo from cells or tissues (Wagner et al. 2011, 2012; Udeshi et al. 2012, 2013) (Fig. 9.2b). For example, Wagner et al. characterized [20,000 ubiquitination sites of [5,200 proteins in murine tissues. "

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    • "Accordingly, the changes in PFK protein levels could be attributed, at least in part, to an increase in gene transcription due to the stabilization of HIF1α by proteasome inhibitors, as HIF1α is a well-known activator of PFK gene transcription during hypoxia [30]. The effects reported by Udeshi et al. [28] after treatment of the cells for 4 h with 5 μM of a less specific proteasome inhibitor, MG132, are not as extensive in the whole proteome but still are highly significant . The expected effects on protein levels by treatment of cells with proteasome inhibitors are time-dependent as transcription, processing, and translation are needed before a change in protein levels can be observed, and 4 h is enough time to observe those effects translated into protein levels. "
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    ABSTRACT: Protein homeostasis, proteostasis, is essential to understand cell function. Protein degradation is a crucial component of the proteostatic mechanisms of the cell. Experiments on protein degradation are nowadays present in many investigations in the field of molecular and cell biology. In the present paper, we focus on the different experimental approaches to study protein degradation and present a critical appraisal of the results derived from steady-state and kinetic experiments using detection of unlabelled and labelled protein methodologies with a proteostatic perspective. This perspective allows pinpointing the limitations in interpretation of results and the need of further experiments and/or controls to establish "definitive evidence" for the role of protein degradation in the proteostasis of a given protein or the entire proteome. We also provide a spreadsheet for simple calculations of mRNA and protein decays for mimicking different experimental conditions and a checklist for the analysis of experiments dealing with protein degradation studies that may be useful for researchers interested in the area of protein turnover.
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